isolation and biochemical characterization of heavy metal resistant bacteria from tannery effluent in chittagong city bangladesh bioremediation viewpoint

Egyptian Journal of Aquatic Research (2017) xxx, xxx–xxx H O S T E D BY National Institute of Oceanography and Fisheries Egyptian Journal of Aquatic Research http://ees.elsevier.com/ejar www.sciencedirect.com Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint Lolo Wal Marzan *, Mehjabeen Hossain, Sohana Akter Mina, Yasmin Akter, A.M Masudul Azad Chowdhury Department of Genetic Engineering and Biotechnology, Faculty of Biological Sciences, University of Chittagong, Chittagong 4331, Bangladesh Received 27 June 2016; revised 21 August 2016; accepted 30 November 2016 KEYWORDS Gemella sp.; Micrococcus sp.; Hafnia sp.; Heavy metal resistance; Degradation capacity; Bioremediation; Characterization Abstract Toxic, mutagenic and carcinogenic heavy metals from tannery industries cause the pollution of agricultural environment and natural water sources This study aims to isolate, investigate and identify naturally occurring bacteria capable of reducing and detoxifying heavy metals (Chromium, Cadmium and Lead) from tannery effluent Three isolates were identified up to genus level based on their morphological, cultural, physiological and biochemical characteristics as Gemella sp., Micrococcus sp and Hafnia sp Among them Gemella sp and Micrococcus sp showed resistance to Lead (Pb), chromium (Cr) and cadmium (Cd), where Hafnia sp showed sensitivity to cadmium (Cd) All isolates showed different MICs against the above heavy metals at different levels Degrading potentiality was assessed using Atomic Absorption Spectrophotometer where Gemella sp and Micrococcus sp showed 55.16 ± 0.06% and 36.55 ± 0.01% reduction of Pb respectively On the other hand, moderate degradation of Cd was shown by Gemella sp (50.99 ± 0.01%) and Micrococcus sp (38.64 ± 0.06%) Heavy metals degradation capacity of Gemella sp and Micrococcus sp might be plasmid mediated, which might be used for plasmid transformation to transfer heavy metal accumulation capability Therefore, identification of three bacteria for their heavy metal resistance and biodegradation capacity might be a base study to develop the production of potential local bioremediation agents in toxic tannery effluent treatment technology Ó 2016 National Institute of Oceanography and Fisheries Hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) * Corresponding author Fax: +880 31 2606145, +880 31 2606014 E-mail addresses: marzan.geb@cu.ac.bd, lmarzancu@yahoo.com (L.W Marzan) Peer review under responsibility of National Institute of Oceanography and Fisheries Introduction Air, water and land which are the essential elements of life are contaminated constantly due to increasing population, rapid urbanization and industrialization (Chhikara and Dhankhar, http://dx.doi.org/10.1016/j.ejar.2016.11.002 1687-4285 Ó 2016 National Institute of Oceanography and Fisheries Hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Please cite this article in press as: Marzan, L.W et al., Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint Egyptian Journal of Aquatic Research (2017), http://dx.doi.org/10.1016/j.ejar.2016.11.002 2008) At present, the bioaccumulation of heavy metals in environment is a major warning to human life (Yigit and Ahmet, 2006; Hooda, 2007) Water pollution caused by industrial wastage, is frequent (Ogedengbe and Akinbile, 2004) by toxic sludge, heavy metals, and solvents as they fall into natural water sources and agricultural environment Heavy metals containing industrial effluent cause health hazards to plants, animals, aquatic life and humans increasing pressures on the flora and fauna (Robin et al., 2012) Among industrial usage of heavy metals, tannery industries use a significant part of it Tannery effluent is highly polluted because it contains imbalance suspended solids, nitrogen, conductivity, sulfate, sulfide and chromium, copper, cadmium and manganese, biological oxygen demand (BOD) and chemical oxygen demand (COD) (Mondal et al., 2005; Zahid et al., 2006) In Bangladesh unprocessed tannery effluents are released into water sources (Favazzi, 2002; Verheijen et al., 1996) Consequently, the elevated concentrations of some heavy metals are found in agricultural soils located in surrounding areas to the tannery industries which exceed the tolerable limit Lead and cadmium which are major contaminants found in the environment, are extremely poisonous to human(s), animals, plants and microbes which can damage cell membranes, alter particularity of enzymes, and destroy the structure of DNA This toxicity is created by the displacement of essential metals from their native binding sites or ligand interactions (Olaniran et al., 2013) Chrome powder and chrome liquor are applied in tanning industry, and are highly toxic heavy metals (Cr6+) which cause water pollution (Sing, 1994), where a lot of (>170000 tons) chromium wastes are released to the surroundings (Kamaludeen et al., 2003) It causes health hazards since it can easily enter biological cell membranes (Chaudhary et al., 2003) Tanned skin-cut wastes (SCW) which are used to produce feeds and fertilizers, are the direct phenomenon of chromium toxicity (Rafiqullah et al., 2008) Hexavalent Chromium (Cr6+) is 100–1000 times more poisonous compared to trivalent (Cr3+) form (Gauglhofer and Bianchi, 1991) So, conversion of Cr6+ is one of the significant mechanisms for microorganisms that can be used for detoxification of chromium Lacking a single waste-water treatment facility, a notorious and substantial ruin of the environment is another fate of concern from such a pivotal industry to sustain a billion-dollar business An excess of such chemicals in the water and soils is harmful for the health of the people crammed into the area (Sunder et al., 2010) The breakthrough toward the sustainable mitigation of this overwhelming problem is nothing but the installation of an appropriate effluent treatment plant in every industry in terms of efficiency, cost effectiveness, simplicity and more importantly it should be environment friendly Due to lack of treatment plants and environment management schemes in most of the tanneries in our country, raw wastes are simply discharged into the environment, causing severe environmental and public health troubles in particular areas Appropriate environmental management is needed (Hasnat et al., 2013) to overcome this hazardous issue and tannery productivity Several microorganisms have developed detoxification and respiration mechanism using heavy metals and thus become resistant to it (Ezaka and Anyanwa, 2011) The isolation and characterization of heavy metal resistant bacteria is significant for its metal accumulation capability along with its resistance capacity In our study the sampling sites are the L.W Marzan et al three nearby surroundings of Madina tannery which is the largest tannery located at Jalalabad area, near Oxygen point in Chittagong, Bangladesh It was established in 1983, and is renowned for manufacturing all kinds of export quality crust and refined leather In the surroundings of Madina tannery, large municipal areas have been observed So, the present study aims to investigate the ability of natural inhabitant bacteria of tannery effluent in reducing and detoxifying of heavy metals (Pb, Cr and Cd) at privileged conditions, where objectives include – isolation of naturally occurring bacteria from tannery effluent, screening of top three isolates as the reducer of Pb, Cr and Cd, characterization of heavy metal resistance, identification of those bacteria up to genus and profiling of their plasmids as a fundamental research to ensure the basis of their resistance in order to use them for detoxification in an incorporated bioremediation scheme Materials and methods Study area and collection of samples Tannery effluent samples were collected from three agricultural and residential sites beside Madina tannery (Fig 1D, Table 1), in labeled pre-sterilized bottles, and cold chain was maintained during shipment to the laboratory in the University of Chittagong Collected samples were preserved at °C before analysis and during experiments Primary screening of heavy metal resistant bacteria For the selective screening of heavy metal resistant bacteria, 300 lg/mL of heavy metal (Lead) incorporated LB (Luria Bertani) agar plates (Peptone 10.00 g/L, yeast extract, 5.00 g/L, NaCl 5.00 g/L, dextrose anhydrate 10.00 g/L and agar 30.00 g/L: pH À7.00) were used and screened by standard pour plate method observed at 37 °C After 24 h of incubation the plates were observed for any kind of development on the culture medium After preliminary screening of effluent samples containing heavy metal degrading isolates, serial dilution was done as Azad et al (2013) to isolate desired bacteria Streak plate technique was followed during isolation Control plates also prepared with LB media without including any heavy metal to make comparison Colonies differing in morphological characteristics were selected, picked, purified and then preserved on different plates for further studies Multiple metal resistance capacity All isolates (S1, S2, S3, S4 and S5) were separately grown on LB agar plates supplemented with Cd, Cr and Pb (300 lg/ mL) at pH 7.0 and 37 °C for 24 h; whereas after incubation the resistance capacity of multiple heavy metals was assessed Relative effects of heavy metal consumption on microbial growth The optimal growth conditions with reference to different amounts of three heavy metals were determined The isolates were grown in a rotary shaker (Wise cubeÒ, Korea) at 150 rpm and pH 7.0, while the temperature was 37 °C in LB broth medium supplemented with different types of heavy Please cite this article in press as: Marzan, L.W et al., Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint Egyptian Journal of Aquatic Research (2017), http://dx.doi.org/10.1016/j.ejar.2016.11.002 Isolation and characterization of heavy-metal resistant bacteria from tannery effluent Figure Study area map indicating Chittagong City (A), Places of Madina Tannery (B,C) and three sampling sites (D) Table Details of effluent sampling location surroundings of Madina tannery Sites 3 Features of Sites Madina Tannery Nearby area of Signal Battalion Archery Range, Chittagong Jalalabad Word no 2, Chittagong Nearby area of Chittagong-RangamatiKhagrachori Highway Latitude Longitude 00 22° 40 06.24 N 22° 400 05.2400 N 91° 810 88.7300 E 91° 810 83.2600 E 22° 390 90.1700 N 91° 810 64.3800 E 22° 390 85.4100 N 91° 810 97.8500 E metals (gradually increasing 100 lg/mL at every time, until it reaches to 1000 lg/mL) separately The optical density (OD) was measured (at k = 600 nm) using UV spectrophotometer (Shimadzu, Japan) After 6–8 h of incubation the effect of heavy metal concentration on their growth was assessed Determination of minimum inhibitory concentration (MIC) To asses MIC, heavy metal resistant selected isolates were grown on heavy metal incorporated media against respective heavy metal; was identified by gently inclining the concentration of the heavy metals (Pb, Cd and Cr) on LB agar plates until the isolates failed to give colonies on the petri plate The starting concentration of the heavy metals was 50 lg/mL and the culture growing on the final concentration was transferred to the higher concentration each time by streaking on the agar plate When the isolates failed to grow on petri plate, MIC was assessed according to standard protocol of European food safety authority (EFSA), Parma, Italy, 2012 Please cite this article in press as: Marzan, L.W et al., Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint Egyptian Journal of Aquatic Research (2017), http://dx.doi.org/10.1016/j.ejar.2016.11.002 L.W Marzan et al Heavy metal biodegradability assay Bacterial isolates were cultured into shake flask containing LB broth medium for one hour in a rotary shaker at 150 rpm, while pH and temperature were maintained 7.0 and 37 °C respectively After optical density is reached at 0.6 (k = 600 nm: for equal enzyme activity), then 100 ppm of sterilized heavy metal (Pb, Cd or Cr) was added separately in every culture flask and again incubated for 24 h at same condition Then total culture was centrifuged (Sigma 2-16KL, Germany) at 5000 rpm for 15 The supernatants were separated and mixed to the double volume of concentrated HNO3 Then those mixtures were heated to 100 °C on a Hotplate Stirrer (Lab tech-Daihan Company) to accomplish acid digestion until the final volume decrease and down to initial supernatant volume Through a filter paper (Whatman 42) the extract was filtered to remove any insoluble material and collected into a volumetric flask and then diluted This extract of total heavy metal reduction was analyzed by Atomic Absorption Spectrophotometer (Shimadzu AA-7000, Japan) and the result was compared with control to calculate heavy metal degradation capacity (%) as follows: % of heavy metal utilized Heavy metal utilized ppmị ẳ 100 Heavy metal added to the LB broth ðppmÞ Heavy metal utilized ppmị ẳ Heavy metal added to the LB broth ppmị À Heavy metal at the end of culture ðppmÞ Phenotypic and biochemical characterization of bacterial isolates The bacterial isolates were characterized based on cultural, morphological and biochemical characteristics as described in the Cowan and Steel’s Manual for the identification of Medical Bacteria (Barrow and Feltham, 1993) For the activities of oxidase, catalase, methyl red, indole production, citrate utilization and carbohydrate (Glucose, Sucrose, Maltose, Xylose and Lactose) utilization, isolates were biochemically analyzed (Barrow and Feltham, 1993) According to Bergey’s Manual of systemic Bacteriology the isolates were provisionally identified up to genus level (Claus and Berkeley, 1986) Statistical analysis Triplicate measurements were done in all the cases during the observation and assessment of bacterial growth incorporated with different levels of heavy metals Data were captured into Microsoft Excel Software, version 2010 which was used to calculate means and standard deviations Student’s t-test was applied to confirm that the observed changes were statistically significant Plasmid DNA extraction Plasmid DNA extractions of heavy metal resistant bacteria were done according to the alkaline lysis method (Sambrook and Russel, 2001) Then visualized it on gel electrophoresis at 1% agarose gel and compared with marker DNA (1kbp sharp DNA Marker- RBC Bioscience) to assess plasmid DNA Results Screening and isolation of heavy metal resistant bacteria Visual observation of growth in heavy metal (Lead) supplemented (300 lg/mL) LB medium after 24 h of incubation indicated that each of the collected sample consists of heavy metal degrading bacteria, as they can grow there by degrading heavy metals After primary screening of the collected samples, serial dilution was conducted to isolate desired bacteria (Table 2) Total twenty bacterial isolates were isolated, among them five isolates (S1, S2, S3, S4 and S5) were selected for further study Comparative analysis for multiple heavy metal degrading ability To ascertain potential heavy metal degrading bacterial isolates, we have conducted growth curve analysis for five isolates in LB broth medium containing heavy metals (Pb, Cd or Cr, separately), and their resistance capacity was assessed (data not shown for S2 or S3) In this experiment, S1 and S4 showed good tolerance capacity against multiple heavy metals Besides, S5 showed better tolerance to Pb and Cr, whereas it showed sensitivity to Cd (Table 3) Relative heavy metals consumption rate on bacterial growth Hundred lg/mL amount of heavy metals (Cd, Cr or Pb, separately) were supplemented in LB broth medium for each isolate, where each heavy metal concentration was gradually increased from100 to 1000 lg/mL The cultures were incubated for 6–8 h and measured for optical density (at k = 600 nm) in UV spectrophotometer to study the relative consumption rate and bacterial resistance against each heavy metal All bacteria showed high tendency to decrease optical density while increasing metal concentration (except Cr) in the medium (Fig 4; Table 3) Then three potential isolates (S1, S4 and S5) have been selected for conducting further bioremediation tests Assessment of MIC against each heavy metal Minimum inhibitory concentration (MIC) for each heavy metal was examined ranging from 50 to 1900 lg/mL It was found that all isolates exhibited resistance to heavy metals MIC of heavy metals showed highest tolerance to Pb, by the selected isolates S1 and S4 (Table 3) Table Total viable cell count Dilution factor of the sample Heavy metal (Pb) incorporated media Control plate (without heavy metal-Pb) Percentage of heavy metal (Pb) resistant bacteria 101 102 103 2.5 Â 102 0.8 Â 103 0.54 Â 104 8.05 Â 102 5.03 Â 103 2.2 Â 104 31.05% 35.78% 24.54% Please cite this article in press as: Marzan, L.W et al., Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint Egyptian Journal of Aquatic Research (2017), http://dx.doi.org/10.1016/j.ejar.2016.11.002 Isolation and characterization of heavy-metal resistant bacteria from tannery effluent Table Morphological and biochemical characteristics, carbohydrate utilization test, heavy metal resistance capacity and MIC of bacterial isolates (Barrow and Feltham, 1993; Claus and Berkeley, 1986) Bacterial Isolates S1 and S4 showed $1.4 times higher (P < 0.01) than S1 On the other hand, S1 showed $7.3 times (P < 0.01), as well as S4 showed $5.5 times higher (P < 0.01) Cd degrading capacity than S5 (Fig 5) S4 S5 Characterization and identification Morphological characteristics Colony color White Milky Gram Nature Positive Cell shape Round Yellowish Brown Positive Round Whitish, not milky Negative Rod Biochemical test results Oxidase Catalase Indole Methyl-Red Citrate + + À À À À + À + À Top three potential heavy metal degrading isolates (S1, S4 and S5) were characterized based on their cultural, morphological and biochemical characteristics (Table 3) Compared with standard description of Bergey’s Manual of determinative bacteriology 9th edition (Bergey et al., 1974; Williams and Wilkins, 1994), the isolates were provisionally identified up to genus level as Gemella sp (S1); Micrococcus sp.(S4) and Hafnia sp (S5) are consistent with past field studies (Claus and Berkeley, 1986) À À À À + Utilization of carbohydrate Glucose + Sucrose Maltose + Xylose Lactose Resistance capacity Pb Cr Cd MIC (lg/mlÀ1) Pb Cr Cd Provisionally Identified Bacteria Claus and Berkeley (1986) + Plasmid DNA extraction + À À + À Only Gemella sp (Fig 6, Lane and 2) and Micrococcus sp (Fig 6, Lane and 4) harbor plasmid, while Hafnia sp did not show any plasmid +++ + ++ +++ +++ ++ Discussion ++ ++ + À 1900 360 1350 Gemella sp 1800 345 1100 Micrococcus sp 1200 300 50 Hafnia sp Being an industrial city, Chittagong is facing pollution problems Heavy metals discharged from leather and other industries, as well as ship breaking yard, pose threat to human population, marine biodiversity as well as agricultural environment Though, huge amounts of heavy metals in our environment cause a devastating effect; no effective remediation technique has still been taken in hand Bioremediation is cost-effective, safe and eco-friendly; can be virtually restored a solution to its pure condition (Liu et al., 1997) So, the major focus of this study is to isolate and identify the major bioremediating bacterial agents that help the natural recovery of surroundings (agricultural and residential environment) and assess their comparative heavy metal remediation capacity to select few isolates that can be a solution for recovering future pollution by untreated heavy metal containing effluent Preliminary screening of the collected samples for heavy metal resistance ability showed that all samples were positively grown utilizing heavy metal (Pb) in their culture media Serial dilutions of all samples yield five (5) distinct isolates from the heavy metal resistant bacterial population based on their morphology (Table 2; Figs and 3) The bacterial isolates were then characterized by morphological, biochemical tests, multiple heavy metal resistance capacity, MIC and comparative heavy metal degradation capacity Identification of bacterial isolates were done (Table 3) according to Bergey’s Manual of Determinative Bacteriology (Barrow and Feltham, 1993; Bergey et al., 1974) Depending on gram staining, two isolates (S1 and S5) were identified as gram-positive and the other one (S4) as gramnegative bacteria (Table 3) by detecting peptidoglycan which is present in a thick layer in bacteria (Burke and Pister, 1986) Micrococcus sp are oxidase-positive, which can be used to distinguish them from other gram positive bacteria like most Staphylococcus sp., which are generally oxidase-negative (Thelwell et al., 1998) In our study, S4 (Micrococcus sp.) was identified as oxidase positive as well as it may be Figure Culture plates (A) with and (B) without heavy metal incorporated media Assessment of heavy metal biodegradation capacity To measure total heavy metal (Pb, Cr or Cd) biodegradation capacity, the treated samples were analyzed by Atomic Absorption Spectrophotometer and compared with control Among the isolates, S1 showed $3 times (P < 0.01) Lead (Pb) degrading ability and S4 showed $2 times higher (P < 0.01) compare to isolate S5 In the case of Cr, S1 showed $1.7 times higher degrading ability (P < 0.05) compare to S5, Please cite this article in press as: Marzan, L.W et al., Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint Egyptian Journal of Aquatic Research (2017), http://dx.doi.org/10.1016/j.ejar.2016.11.002 L.W Marzan et al Figure Pure cultures of five (S1, S2, S3, S4, S5) bacterial isolates Micrococcus luteus, since it produced yellow to brown colonies (Table 3) on growth medium compared to the red colony of Micrococcus roseus Gram staining, oxidase tests and carbohydrate utilization studies showed (Table 3) similarities of S1 with Gemella sp., which is gram positive, oxidase negative and utilizes all carbohydrates (Nucifora et al., 1989) A coliform is an aerobic or facultative anaerobic rod and gram negative, which as identified by IMViC test can produces gas from lactose within 48 h These coliforms indicate fecal contamination in the previous study, where it has been done to confirm the presence of coliform bacteria in industrial effluent samples (Malik and Jaiswal, 2010) Our study found S5 is coliform bacteria and identified as Hafnia sp (data not shown) More specific study with some other industrial water effluents, established the presence of Hafnia alvei in tannery effluent, which is very much deleterious to our environment (Stackebrandt et al., 1982) The multi-metal resistance capacity approached two bacterial isolates Gemella sp and Micrococcus sp are highly resistant to Pb and Cd compared to Hafnia sp (Table 3) Besides, Cd is a more lethal heavy metal for Hafnia sp (Table 3) Upon above experiments, the resistance level Pb > Cd > Cr showed for Gemella sp and Micrococcus sp It was also reported that the tolerant levels of heavy metal for sewage bacteria Pseudomonas aeruginosa, Acinetobacter resistance, Proteus vulgaris were shown to be Pb > Cd > Cr (Powers and Latt, 1977) Relative effects of bacterial growth in presence of heavy metal(s) in different concentrations (100–1000 lg/mL) were studied and it was observed that bacterial growth is concentration dependent, since it showed decreasing optical density (at k = 600 nm) in accordance with the increasing heavy metal concentration (Fig 4) Besides, Hafnia sp shows sensitivity to Cd as well as their resistance capacity against Pb and Cr are also lower compared to other bacteria Interestingly, Gemalla sp and Micrococcus sp show resistance and tolerance capacity against Pb and Cr On the other hand the result of Cd resistance is insignificant (Fig 4C) Minimum inhibitory concentration (MIC) is the lowest concentration at which the isolate is completely suppressed (as demonstrated by the absence of visible bacterial growth) is recorded In this study order of MICs for the isolates S1 and S4 was found to be Pb > Cd > Cr and Pb > Cr > Cd for the isolate S5 (Table 3) Gemella sp is resistant against Cd with MIC of 128 lg/mL, Cr with MIC of 1024 lg/mL has been recently shown by Ashour et al (2011) Gemella sp was characterized in our study with MIC against Cd 1350 lg/mL, Cr 360 lg/mL as well as Pb 1900 lg/mL (Table 3) Janda (2006) demonstrated that 13 bacteria are resistant to heavy metals (Zn, Pb, Cr, Cd); where Micrococcus luteus was found to be the most multiple heavy metals resistant Our study found the multiple heavy metal tolerance capacity for Micrococcus sp.; with MIC against Pb (1800 lg/mL), Cr (345 lg/mL) and Cd (1100 lg/mL) being the second highest capacity (Table 3) Among three characterized bacteria, Hafnia sp was identified here as the lowest capacity of Pb and Cr reducing bacteria, was also sensitive to Cd and is similar to the recent studies by Fakhruddin et al (2009) and Resende and Silva (2012) The isolates measured by Atomic Absorption Spectrophometer; Gemella sp and Micrococcus sp showed considerable degradation of Pb which were 55.16 ± 0.06% and 36.55 ± 0.01%, respectively On the other hand Hafnia sp shows very low degradation capacity (18.28 ± 0.06%) Degradation of Cd by Gemella sp and Micrococcus sp showed 50.99 ± 0.01% and 38.64 ± 0.06% respectively, where Hafnia sp was Cd sensitive But degradation of Cr was moderate for all isolates which showed 6.14 ± 0.24, 8.42 ± 0.02 and 3.69 ± 0.2% for Gemella sp., Micrococcus sp and Hafnia sp respectively It might be due to existence here of hexavalent Chromium (Cr6+), which is known to be 100–1000 times more toxic than trivalent (Cr3+) form (Gauglhofer and Bianchi, 1991) So, it is found here, conversion of Cr6+ may not be much easier with these three bacteria, where waste water detoxification mechanisms by microorganism are an important factor Bacterial plasmids encode resistance systems for toxic metal ions are inherited by plasmids in many bacteria (Silver and Phung, 1996) Recent study shows heavy metal resistance capacity either plasmid mediated or chromosomal DNA Please cite this article in press as: Marzan, L.W et al., Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint Egyptian Journal of Aquatic Research (2017), http://dx.doi.org/10.1016/j.ejar.2016.11.002 Isolation and characterization of heavy-metal resistant bacteria from tannery effluent Figure Optical density (k = 600 nm) was measured at UV Spectrophotometer (Shimadzu, Japan) after 6–8 h incubation in LB broth medium incorporated with heavy metals as Pb (A), Cr (B) and Cd (C) to observe relative heavy metal consumption rate on the growth of bacterial isolates mediated (Virender et al., 2010) For determination of genetic basis for metal resistance, plasmid profiling is important Plasmid DNA extraction of three bacterial isolates having biodegradation capacity was assessed to understand whether their heavy-metal resistance capacity is plasmid DNA or chromosomal DNA mediated In our study, two strains Gemella sp and Micrococcus sp showed plasmid DNA, while Hafnia sp does not harbor plasmid (Fig 6) Probably the high degrading capacity of Gamella sp and Micrococcus sp can be the reason for their plasmid retaining ability (Ghosh et al., 1997), where Hafnia sp has lower degrading ability without plasmid DNA In bacteria, the heavy metal resistant genes are located either on the bacterial chromosome or in the plasmids or on both (Nies and Brown, 1997) According to Malik (2004), Cd and Cr resistant genes are present in plasmid DNA but Pb resistance gene is located on chromosomal DNA of Enterobacteria In this way, chromosomal gene might be responsible for this kind of lower degrading capability but more usually conferring resistance are located on plasmid (Woertz and Mergeny, 1997) Although this fundamental study will support for plasmid curing, transformation and evaluation of heavy metal resistance can pave way for the genetic basis of heavy metal resistant mechanism Plasmid mediated heavy metal resistance is important for further transformation study, which will render any heavy metal sensitive bacteria (recipient) into being heavy metal resistant bacteria (Mergeay et al., 2003; Vaijiheh and Naser, 2003) Further study of the effects of different supplements and conditions in their growth is needed to identify their efficiency as bioremediation agents, where optimization of pH, temperature, and incubation time can influence metal resistance capacity (Shivakumar et al., 2014) To make it usable for local farmers in their paddy fields and hatcheries, this is the base study to develop three biological Please cite this article in press as: Marzan, L.W et al., Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint Egyptian Journal of Aquatic Research (2017), http://dx.doi.org/10.1016/j.ejar.2016.11.002 L.W Marzan et al Figure Heavy metals’ (Pb, Cr or Cd) degradation capacity (%) by each bacterial isolates Triplicate measurements were done and compared with control in each case Error bars indicate ±SD Total heavy metal reduction was analyzed by Atomic Absorption Spectrophotometer M 10000bp 8000bp 7000bp 6000bp 5000bp 4000bp 3000bp 2800 bp (approx ) 2000bp 1900 bp (approx ) 1000bp 500bp Figure Lane and show plasmid of Gemella sp.; and show plasmid of Micrococcus sp.; and shows no plasmid band of Hafnia sp agents with resistance and degradation capacity, focusing tannery effluent pollution, might be helpful to formulate and develop local production of bioremediation agents for human, agricultural and aquatic environmental cleaning Conclusion In this study, twenty samples were collected from tannery industrial surroundings in three contaminated sites and their heavy metal degrading potentiality was assessed From those samples, five (5) bacterial isolates have been selected The isolates were subjected to study multiple metal resistance capacity, growth curve analysis on different concentrations of heavy metals, MIC and heavy metal biodegradability analysis to select the best candidates that might be further used for bioremediation of heavy metal pollutants Depending on various tests for biochemical characterization; we identified them as Gemella sp Micrococcus sp and Hafnia sp All the results presented in this study support the concept that three bacteria (Gemella sp Micrococcus sp and Hafnia sp.) had significant bioremediation potentiality which might be used to formulate bioremediation agents to detoxify tannery effluents at industrial surroundings in the natural environments in Bangladesh Conflict of interest No conflict of interest influenced in this research Authors’ contribution LWM conceived and designed the project and MH carried out the laboratory experiments LWM, SAM and YAR prepared the manuscript LWM supervised and interpreted the results All authors read and approved the final manuscript Please cite this article in press as: Marzan, L.W et al., Isolation and biochemical characterization of heavy-metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint Egyptian Journal of Aquatic Research (2017), http://dx.doi.org/10.1016/j.ejar.2016.11.002 Isolation and characterization of heavy-metal resistant bacteria from tannery effluent Acknowledgement The authors are grateful to Research and Publication Office, University of Chittagong, Chittagong-4331, Bangladesh for partial financial assistance (Memo No 5628/2015; fiscal year 2014–2015) The authors are pleased to mention about the fruitful suggestions for the grammar check from Mahira Taj References Ashour, M.S., Mansy, M.S., Eissa, M.E., 2011 Microbiological environmental monitoring in pharmaceutical facility Egypt Acad J Biol Sci 3, 63–74 Azad, A.K., Nahar, A., Hasan, M.M., Islam, K., Azim, 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from tannery. .. article in press as: Marzan, L.W et al., Isolation and biochemical characterization of heavy- metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint. .. article in press as: Marzan, L.W et al., Isolation and biochemical characterization of heavy- metal resistant bacteria from tannery effluent in Chittagong city, Bangladesh: Bioremediation viewpoint